Multiplex Targeted Amplification Using Flap Nuclease

ABSTRACT

Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5′ or 3′ flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5′ and 3′ flaps are generated. The flaps are cleaved using 5′ or 3′ flap endonucleases or 3′ to 5′ exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.60/885,333, filed Jan. 17, 2007 and 60/887,546 filed Jan. 31, 2007,which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention is related to methods for amplifying a plurality ofspecific targets in a multiplex amplification reaction.

BACKGROUND OF THE INVENTION

Over the past two decades, the in vitro amplification of specificnucleic acids has become an essential tool for molecular biologists.More recently, multiplexed amplification, in which a plurality ofnucleic acid sequences are amplified in a single reaction, Chamberlainet al., Nucl. Acid Research 16(23):11141-1156 (1988); U.S. Pat. No.5,582,989, has become increasingly important. For example, multiplexedamplification, particularly multiplexed polymerase chain reaction (PCR),has been used to provide genetic fingerprints of infectious diseaseorganisms. Other applications, such as multiplex SNP genotyping andvariation scanning (for example, by mismatch repair detection), alsogreatly benefit from PCR multiplexing.

In its original implementation, multiplex PCR reactions include aspecific primer pair for each locus to be amplified. These approacheshave been plagued with problems, however, including uneven or failedamplification of some templates (especially those having GCrich-sequences), preferential amplification of other templates, poorsensitivity and specificity, poor reproducibility, and the generation ofspurious amplification products (Henegariu et al., BioTechniques 23(3):504-511 (1997); Markoulatos et al., J. Clin. Lab. Anal. 16: 47-51(2002)).

Various modifications to the original approach have been developed inefforts to minimize these problems. Among these modifications arechanges to the reaction conditions, including adjustment of primerconcentrations, MgCl₂ and dNTP concentrations, changes in PCR bufferconcentrations, balance between MgCl₂ and dNTP concentrations, amountsof template DNA and Taq DNA polymerase, extension and annealing time andtemperature, and the addition of adjuvants (Henegariu et al.,BioTechniques 23(3): 504-511 (1997); Markoulatos et al., J. Clin. Lab.Anal. 16: 47-51 (2002)). Other strategies used include subcyclingtemperatures between high and low temperatures below the denaturationtemperature, used during the annealing and elongation steps (U.S. Pat.No. 6,355,422), and the use of one sequence-specific primer and onecommon primer (Broude et al., Proc. Natl. Acad. Sci. USA 98, 206-211(2001))

The intractability of GC-rich sequences to multiplex PCR has also beenaddressed by a method in which addition of betaine and dimethylsulfoxide(DMSO) to the PCR reaction mix is said to allow more uniformamplification from a heterogeneous population of DNA molecules, many ofwhich were GC-rich (Baskaran et al., Genome Research 6: 633-638 (1996)).

Yet other approaches alter the primers. In one such effort, chimericoligonucleotides are used as primers: the oligonucleotides include a 3′domain that is complementary to template, conferring templatespecificity, and a 5′ domain that is noncomplementary to template; the5′ domain includes a sequence used to prime extension in rounds of PCRamplification subsequent to the first. In this latter scheme, however,the cycles of amplification following the first amplify whatever productis generated in the first cycle, whether correct or erroneous. Thus,while the technique allows for more uniform amplification, it does notaddress the problem of spurious products.

In an analogous approach designed to clone the shared components in twocomplex samples, Brookes et al., Human Molec. Genetics 3(11):2011-2017(1994), ligate primers to template ends generated by restrictionfragment digestion. None of the above-mentioned approaches, however,fully solves the problems associated with multiplex PCR. Thus, there isa continuing need in the art for a method that allows the specific anduniform amplification of multiple nucleic acid sequences in a singlereaction, without the generation of spurious products.

Multiplex targeted genome amplification allows simultaneous generationof many targets in the same tube for cost-effective genotyping,sequencing or resequencing. The most powerful targeted amplification hasbeen the polymerase chain reaction (PCR). Traditional multiplex PCR hasbeen used to amplify two or more targets by putting multiple pairs ofprimers simultaneously in the same reaction. However, due to exponentialincrease in primer-dimer interaction when more pairs of primers areincluded as well as unequal amplification rates among differentamplicons, the multiplexing level of this traditional scheme istypically efficient for up to about 20-plex, often with individualprimer concentrations requiring adjustment. This scheme has foundapplications in multiplex real time PCR or microsatelite amplificationand commercial kits, for example, from Qiagen, are available.

Current amplification methods range from non-specific amplification ofthe entire genome, for example, whole genome amplification (WGA) methodssuch as MDA, to highly targeted PCR amplification of a few or a singleselected region of, for example, a few kb. Methods that result inamplification of a reproducible subset of a genome, for example, theAffymetrix whole genome sampling assay (WGSA) may also be used toamplify genomic material for downstream analysis. The WGA methodsgenerally result in a non-selective amplification of the entire genome.The WGSA method results in amplification of a selected subset of thegenome, the subset being defined by the restriction enzyme or enzymesused for cutting the DNA prior to adapter-mediated PCR amplification.Other methods that allow targeted amplification of large numbers ofspecific targets include, for example, the With whole genomeamplification methods being applied to amplifying the whole human genome(a few billion bp) at one end and PCR in targeting a few kb sequences inthe other end, there is a need to have a strategy amplifying 1-100million by that can cover exons and promoter regions of most or all thefunctional genes.

Attempts have been made over the years since the invention of PCR toincrease the multiplex level of PCR. Some of the strategies includetwo-stage PCR with universal tails (Lin Z et al., PNAS 93: 2582-2587,1996; Brownie J. et al., Nucleic Acids Res. 25: 3235-3241, 1997),solid-phase multiplex PCR (e.g., Adams and Kron, U.S. Pat. No.5,641,658; Shapero et al., Genome Res. 11: 1926-1934, 2001), multiplexedanchored runoff amplification (MARA, Shapero et al., Nucleic Acid Res.32: e181, 2004 and U.S. Pat. No. 7,108,976), PCR with primers designedby a special bioinformatical tool (Wang et al., Genome Res. 15: 276,2005), selector-guided multiplex amplification (Dahl F et al., NucleicAcids Res. 33: e71, 2005), and dU probe-based multiplex PCR after commonoligo addition (Faham M and Zheng J, U.S. Pat. No. 7,208,295 and Faham Met al., PNAS 102: 14717-14722, 2005). However, most of above strategiesare either work most efficiently at about 100 to 1000-plex, or sufferlow efficiency, with the exception of the last two strategies that arepotentially scalable to over 10,000-plex (or over a million bp). Themethod of Dahl et al. requires synthesis of long oligo probes(usually >80 bases) and the method of Faham et al. requires synthesis ofdU probes by PCR for each target (Faham M et al., 2005). Multiplex PCRmethods are also disclosed in U.S. Patent publication Nos. 20030104459.See also, Nilsson et al., Trends. Biotechnol. 24(2):83-8, 2006 andStenberg et al., NAR 33(8):e72, 2005.

SUMMARY OF THE INVENTION

Methods for multiplex amplification are disclosed. In general themethods include fragmentation of targets, circularization of targetsusing juxtaposition of the ends of the target using a splintingcircularization probe and ligation of the ends of the targets.

In one embodiment the fragment ends are of known sequence and thecircularization probe is designed to juxtapose the ends so that they canbe ligated.

In another embodiment the circularization probe hybridizes to the targetso that a 5′ flap is formed and a 3′ single base overhang. A 5′ flapnuclease is used to remove the 5′ flap and to generate juxtaposed endsfor ligation.

In another embodiment a 3′ flap structure is formed by hybridization ofthe circularization probe and a 3′ flap nuclease is used to remove the3′ flap and the ends are ligated. DNA polymerase and a subset of dNTPsmay be added.

In another embodiment both a 5′ and a 3′ flap are formed and a 3′ to 5′exonuclease and a 5′ flap nuclease are used to remove the flaps so thatthe resulting juxtaposed ends can be ligated using a ligase.

The resulting circular targets can be amplified using rolling circleamplification and the amplified targets can be analyzed, for example, byhybridization to an array of probes.

A collection of more than 100, 1000, 10,000 or 50,000 different sequencetargets may be amplified in a single multiplex reaction and theamplification products may be analyzed for genotype, copy number,methylation, translocation, or sequence, for example. Kits forcircularization are also contemplated. The kits may contain one or moreof the following: a collection of more than 100, 1,000, 10,000 or 50,000circularization probes, a 3′ flap nuclease, a 5′ flap nuclease, aligase, a DNA polymerase, one or more dNTPs, primers for RCA, a Phi29DNA polymerase and a 3′ to 5′ exonuclease. In some aspects computersoftware is used to design the circularization probes based on thefragmentation method, the target genome and the target sequences.

In a preferred aspect a kit that includes at least 100, 1000, 10,000 or50,000 different circularization probes for amplification of a pluralityof target sequences is disclosed. The kit preferably also includescircularization probes, including one for each target to be amplified, aligase and a flap endonuclease that may be selected from a 5′ flapendonuclease and a 3′ flap endonuclease, a 3′ to 5′ exonuclease or a DNApolymerase and one or more dNTPs.

Methods for multiplex amplification of selected targets are alsodisclosed. In one embodiment a uracil containing probe (dU probe) isgenerated for each target sequence to be amplified. The DNA sample fromwhich the targets are to be amplified is fragmented to generate targetfragments that contain the target sequences. The target fragments alsocontain extra sequence at either the 5′ end, the 3′ end or at both the5′ and 3′ ends. The target fragments are hybridized to the dU probes,which also contain flanking common sequences at the 5′ and 3′ ends. Whenthe target fragments are hybridized to the dU probes 5′ or 3′ flaps arecreated.

In one embodiment a 5′ flap endonuclease is used to remove a 5′ flap. Inanother embodiment a 3′ flap endonuclease is used to remove a 3′ flap, aDNA polymerase and one or more dNTPs may be included to fill any gapscreated. After flap removal common oligonucleotide sequences are ligatedto the target sequences at both the 5′ and 3′ ends and the targetsequences are amplified using primers to the common sequences.

The dU probes are typically digested prior to amplification of thetarget sequences by treatment with UDG and heat or an AP endonuclease.

In one aspect a 3′ to 5′ exonuclease is used to remove 3′ flaps.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention willbe apparent upon consideration of the following detailed descriptiontaken in conjunction with the accompanying drawings, in which likecharacters refer to like parts throughout, and in which:

FIG. 1 shows a schematic of four different embodiments of a multiplexamplification method.

FIG. 2 shows an example of removal of double flaps for ligation. Theexample is the human integrin alpha 6 gene (ITGA6) exon 2.

FIG. 3 shows a schematic of four different embodiments of a multiplexamplification method.

FIG. 4 Taq Polymerase and FEN1 specifically cleave the 5′ flap to createnicks for ligation.

FIG. 5 shows gel separation of the products of removal of a 3′ flap bySso XPF/PCNA mix.

FIG. 6 shows specific amplification of 355 targets from a normal sampleand an FFPE sample.

FIG. 7 shows array results of a dU-based multiplex PCR from normalgenomic DNA.

FIG. 8 shows a comparison of array results from FFPE samples and normalsamples after capture.

DETAILED DESCRIPTION OF THE INVENTION a) General

Reference will now be made in detail to exemplary embodiments of theinvention. While the invention will be described in conjunction with theexemplary embodiments, it will be understood that they are not intendedto limit the invention to these embodiments. On the contrary, theinvention is intended to cover alternatives, modifications andequivalents, which may be included within the spirit and scope of theinvention.

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being, but may also be otherorganisms including, but not limited to, mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. PatentPub. No. 20050074787, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974,5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683,5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832,5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070,5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164,5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555,6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos.PCT/US99/00730 (International Publication No. WO 99/36760) andPCT/US01/04285 (International Publication No. WO 01/58593), which areall incorporated herein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. patentapplication Ser. No. 10/442,021, U.S. Patent Publication No. 20030036069and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460,6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Pat.Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, for example, PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188,and 5,333,675, each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No.09/513,300, which are incorporated herein by reference.

Methods are disclosed for identifying chromosomal gains and losses athigh resolution using high-density microarray genotyping methods such aswhole genome sampling analysis (WGSA) (see, Kennedy et al. (2003), NatBiotechnol, Vol., pp. 1233-1237, U.S. Pat. No. 6,361,947, U.S. PatentPublication Nos. 20030025075, 20020142314, 20040146890, 20030186279,20040072217, 20030186280, and 20040067493 and U.S. patent applicationSer. No. 10/442,021). WGSA simultaneously genotypes more than 10,000SNPs in parallel by allele-specific hybridization to perfect match (PM)and mismatch (MM) probes synthesized on an array. Methods forchromosomal copy number analysis using the Affymetrix Mapping 10K arrayin combination with WGSA, have also been reported in Bignell et al.Genome Res. 14:287-295 (2004) and Huang et al., Hum Genomics 1:287-299(2004). Similar analysis using the Affymetrix Mapping 100K array hasalso been reported in Slater et al., Am. J. Hum. Genet. 77:709-726(2005).

The Whole Genome Sampling Assay (WGSA) reduces the complexity of anucleic acid sample by amplifying a subset of the fragments in thesample. A nucleic acid sample is fragmented with one or more restrictionenzymes and an adapter is ligated to both ends of the fragments. Aprimer that is complementary to the adapter sequence is used to amplifythe fragments using PCR. During PCR fragments of a selected size rangeare selectively amplified. The size range may be, for example, 400-800or 400 to 2000 base pairs. Fragments that are outside the selected sizerange are not efficiently amplified.

The fragments that are amplified by WGSA may be predicted by in silicodigestion and an array may be designed to genotype SNPs that arepredicted to be amplified. Genotyping may be done by allele specifichybridization with probes that are perfectly complementary to individualalleles of a SNP. A set of probes that are complementary to the regionsurrounding each SNP may be present on the array. Perfect match probesare complementary to the target over the entire length of the probe.Mismatch probes are identical to PM probes except for a single mismatchbase. The mismatch position is typically the central position so for a25 base probe the mismatch is position 13.

The methods may be combined with other methods of genome analysis andcomplexity reduction. Other methods of complexity reduction include, forexample, AFLP, see U.S. Pat. No. 6,045,994, which is incorporated hereinby reference, and arbitrarily primed-PCR (AP-PCR) see McClelland andWelsh, in PCR Primer: A laboratory Manual, (1995) eds. C. Dieffenbachand G. Dveksler, Cold Spring Harbor Lab Press, for example, at p 203,which is incorporated herein by reference in its entirety. Additionalmethods of sample preparation and techniques for reducing the complexityof a nucleic sample are described in Dong et al., Genome Research 11,1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592, 6,458,530 and U.S.Patent Publication Nos. 20030039069, 20050079536, 20030096235,20030082543, 20040072217, 20050142577, 20050233354, 20050227244,20050208555, 20050074799, 20050042654 and 20040067493, which areincorporated herein by reference in their entireties.

The design and use of allele-specific probes for analyzing polymorphismsis described by e.g., Saiki et al., Nature 324, 163-166 (1986);Dattagupta, EP 235,726, and WO 89/11548. Allele-specific probes can bedesigned that hybridize to a segment of target DNA from one individualbut do not hybridize to the corresponding segment from anotherindividual due to the presence of different polymorphic forms in therespective segments from the two individuals. Hybridization conditionsshould be sufficiently stringent that there is a significant differencein hybridization intensity between alleles, and preferably anessentially binary response, whereby a probe hybridizes to only one ofthe alleles.

Methods for determining copy number using high density SNP genotypingarrays using the Affymetrix 10K SNP genotyping array and the 100KMapping Set are disclosed. The methods should also be useful forestimating copy number along with a higher density genotyping array,such as the 500K Mapping Set. The 10K array and the 100K array set use aWGSA target preparation scheme in which single primer PCR amplificationof specific fractions of the genome is carried out. The 100K WGSA methoduses two separate restriction enzymes that each generates a complexityfraction estimated to be about 300 Mb. The 10K array uses a singlerestriction enzyme and generates a sample with less than 300 Mbcomplexity. Both arrays have been shown to genotype SNPs, with callrates, reproducibility, and accuracy greater than 99%, 99.7%, and 99.7%respectively (Matsuzaki et al. Nat Methods 1:109-111, 2004).

The term “WGSA (Whole Genome Sampling Assay) Genotyping Technology”refers to a technology that allows the genotyping of thousands of SNPssimultaneously in complex DNA without the use of locus-specific primers.WGSA reduces the complexity of a nucleic acid sample by amplifying asubset of the fragments in the sample. In this technique, a nucleic acidsample is fragmented with one or more restriction enzyme of interest andadaptors are ligated to the digested fragments. A single primer that iscomplementary of the adaptor sequence is used to amplify fragments of adesired size, for example, 400-800, 400-2000 bps, using PCR. Fragmentsthat are outside the selected size range are not efficiently amplified.The processed target is then hybridized to nucleic acid arrayscomprising SNP-containing fragments/probes. WGSA is disclosed in, forexample, U.S. Patent Publication Nos. 20040185475, 20040157243 (also PCTApplication published as WO04/044225), 20040146890, 20030186279,20030186280, 20030232353, and 20040067493, and U.S. patent applicationSer. Nos. 10/442,021 and 10/646,674, each of which is herebyincorporated by reference in its entirety for all purposes.

Given the millions of SNPs that are estimated to exist and the largesubset already in databases, there is a need to prune this number downto a number that will fit on a few microarrays at current feature sizes.Applications of microarray for SNP genotyping have been described ine.g., a number of U.S. patents and patent applications, including U.S.Pat. Nos. 6,300,063, 6,361,947, 6,368,799 U.S. patent application Ser.No. 10/442,021 and US Patent Publication Nos. 20040067493, 20030232353,20030186279, 20050260628, and 20030186280, all incorporated herein byreference in their entireties for all purposes. Methods and arrays forsimultaneous genotyping of more than 10,000 and more than 100,000 SNPshave also been described for example in Kennedy et al. (2003) Nat.Biotech. 21:1233-7, Matsuzaki et al., (2004) Genome Res. 14(3): 414-425,and Matsuzaki et al (2004) Nature Methods, Vol 1, 109-111, allincorporated herein by reference in their entireties for all purposes.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 which is incorporatedherein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) Alves and Carr Nucleic Acid Res 16:8723,(1988) and Barringer et al. Gene 89:117 (1990)), transcriptionamplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)and WO88/10315), self-sustained sequence replication (Guatelli et al.,Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selectiveamplification of target polynucleotide sequences (U.S. Pat. No.6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR)(U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction(AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid basedsequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818,5,554,517, and 6,063,603, each of which is incorporated herein byreference). Other amplification methods that may be used are describedin, U.S. Pat. Nos. 6,582,938, 5,242,794, 5,494,810, 4,988,617, each ofwhich is incorporated herein by reference. Amplification may also be bymultiple displacement amplification. For a description of multipledisplacement assay, see for example Lasken and Egholm, TrendsBiotechnol. 2003 21(12):531-5; Barker et al. Genome Res. 2004 May;14(5):901-7; Dean et al. Proc Natl Acad Sci USA. 2002; 99(8):5261-6; andPaez, J. G., et al. Nucleic Acids Res. 2004; 32(9):e71. Multiplex PCRprocedures are also disclosed in Shuber et al. Genome Res 5(5):488-93(1995), Brinson et al. Introduction to PCR/OLA/SCS, a multiplex DNAtest, and its application to cystic fibrosis. Genet Test. 1997;1(1):61-8,

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Patent Pub. Nos. 20030096235, 20030082543 and 20030036069.

In preferred embodiments large scale mapping of disease loci may beperformed using a fixed panel of SNPs that interrogate the entire genomeat a selected resolution. Arrays capable of interrogating fixed SNPpanels are available from Affymetrix and include, for example, theMapping 10K array, the Mapping 100K array set (includes 2 50K arrays)and the Mapping 500K array set (includes two ˜250K arrays). These arraysand array sets interrogate more than 10,000, 100,000 and 500,000different human SNPs, respectively. The perfect match probes on thearray are perfectly complementary to one or the other allele of abiallelic SNP. Each SNP is interrogated by a probe set comprising 24 to40 probes. The perfect match probes in a probe set are each different,varying in, for example, the SNP allele, the position of the SNPrelative to the center of the probe and the strand targeted. The probesare present in perfect match-mismatch pairs. The SNPs interrogated by amapping array or array set are spaced throughout the genome withapproximately equal spacing, for example, the SNPs in the 10K array areseparated by about 200,000 base pairs. The median physical distancebetween SNPs in the 500K array set is 2.5 kb and the average distancebetween SNPs is 5.8 kb. The mean and median distance between SNPs willvary depending on the density of SNPs interrogated. Methods for usingmapping arrays see, for example, Kennedy et al., Nat. Biotech.21:1233-1237 (2003), Matsuzaki et al., Genome Res. 14:414-425 (2004),Matsuzaki et al., Nat. Meth. 1:109-111 (2004) and U.S. Patent Pub. Nos.20040146890 and 20050042654. Selected panels of SNPs can also beinterrogated using a panel of locus specific probes in combination witha universal array as described in Hardenbol et al., Genome Res.15:269-275 (2005) and in U.S. Pat. No. 6,858,412. Universal tag arraysand reagent kits for performing such locus specific genotyping usingpanels of custom molecular inversion probes (MIPs) are available fromAffymetrix.

Computer implemented methods for determining genotype using data frommapping arrays are disclosed, for example, in Liu, et al.,Bioinformatics 19:2397-2403 (2003), Rabbee and Speed, Bioinformatics,22:7-12 (2006), and Di et al., Bioinformatics 21:1958-63 (2005).Computer implemented methods for linkage analysis using mapping arraydata are disclosed, for example, in Ruschendorf and Nurnberg,Bioinformatics 21:2123-5 (2005) and Leykin et al., BMC Genet. 6:7,(2005). Computer methods for analysis of genotyping data are alsodisclosed in U.S. Patent Pub. Nos. 20060229823, 20050009069,20040138821, 20060024715, 20050250151 and 20030009292.

Methods for analyzing chromosomal copy number using mapping arrays aredisclosed, for example, in Bignell et al., Genome Res. 14:287-95 (2004),Lieberfarb, et al., Cancer Res. 63:4781-4785 (2003), Zhao et al., CancerRes. 64:3060-71 (2004), Nannya et al., Cancer Res. 65:6071-6079 (2005)and Ishikawa et al., Biochem. and Biophys. Res. Comm., 333:1309-1314(2005). Computer implemented methods for estimation of copy number basedon hybridization intensity are disclosed in U.S. Patent Pub. Nos.20040157243, 20050064476, 20050130217, 20060035258, 20060134674 and20060194243.

In another embodiment target amplification by the disclosed methods isused for array-based sequencing applications. The sequence of a nucleicacid may be compared to a known reference sequence by hybridization toan array of probes that detects all possible single nucleotidevariations in the reference sequence. Such arrays, known as resequencingarrays, are commercially available from Affymetrix, Inc. and have beendescribed, for example, see Cutler, D. J. et al., Genome Res. 11(11),1913-25, 2001. During sample preparation for resequencing analysistarget sequences are amplified. Amplification may be, for example, byPCR amplification using pairs of primers that are specific for segmentsof the target to be analyzed. Long range PCR may be used. Stranddisplacing amplification methods may also be used, for example, thetarget may be amplified by long range amplification using a stranddisplacing enzyme such as Phi 29 or Bst DNA polymerase. Resequencingarrays may be used to analyze both strands of 30 kb or more and 300 kbor more to detect polymorphisms in the sample sequence compared to areference sequence.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in U.S. Pat. Nos. 6,852,938, 5,242,794, 5,494,810, and4,988,617 each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and6,872,529 and U.S. Patent Publication Nos. 20030036069, 20030096235 and20030082543. Additional methods of using a genotyping array aredisclosed, for example, in U.S. patent application Ser. No. 10/442,021,and U.S. Patent Publication Nos. 20040146883, 20030186280, 20030186279,20040067493, 20030232353, 20060292597, 20050233354, 20050074799 and20040185475.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832, 5,631,734, 5,834,758, 5,936,324,5,981,956, 6,025,601, 6,141,096, 6,185,030, 6,201,639, 6,218,803, and6,225,625 in U.S. Patent Pub. No. 20040012676 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758, 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent Pub. Nos.20040012676 and 20050059062 and in PCT Application PCT/US99/06097(published as WO99/47964), each of which also is hereby incorporated byreference in its entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes, etc. The computer-executable instructions may be writtenin a suitable computer language or combination of several languages.Basic computational biology methods are described in, for example,Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Patent Pub. Nos. 20030097222, 20020183936,20030100995, 20030120432, 20040002818, 20040126840, and 20040049354.

b) Definitions

The term “array” as used herein refers to an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formats,for example, libraries of soluble molecules; libraries of compoundstethered to resin beads, silica chips, or other solid supports.

The term “complementary” as used herein refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%. Alternatively,complementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically,selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind noncovalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan 1 M and a temperature of at least 25° C. For example, conditions of5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations. For stringent conditions, see, for example, Sambrook,Fritsche and Maniatis. “Molecular Cloning A laboratory Manual” 2^(nd)Ed. Cold Spring Harbor Press (1989) which is hereby incorporated byreference in its entirety for all purposes above.

The term “hybridization conditions” as used herein will typicallyinclude salt concentrations of less than about 1M, more usually lessthan about 500 mM and preferably less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and preferably in excessof about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone.

The term “hybridization probes” as used herein are oligonucleotidescapable of binding in a base-specific manner to a complementary strandof nucleic acid. Such probes include peptide nucleic acids, as describedin Nielsen et al., Science 254, 1497-1500 (1991), and other nucleic acidanalogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (for example, total cellular)DNA or RNA.

The term “label” as used herein refers to a luminescent label, a lightscattering label or a radioactive label. Fluorescent labels include,inter alia, the commercially available fluorescein phosphoramidites suchas Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (ABI). SeeU.S. Pat. No. 6,287,778.

The term “oligonucleotide” or sometimes refer by “polynucleotide” asused herein refers to a nucleic acid ranging from at least 2, preferableat least 8, and more preferably at least 20 nucleotides in length or acompound that specifically hybridizes to a polynucleotide.Polynucleotides of the present invention include sequences ofdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may beisolated from natural sources, recombinantly produced or artificiallysynthesized and mimetics thereof. A further example of a polynucleotideof the present invention may be peptide nucleic acid (PNA). Theinvention also encompasses situations in which there is a nontraditionalbase pairing such as Hoogsteen base pairing which has been identified incertain tRNA molecules and postulated to exist in a triple helix.“Polynucleotide” and “oligonucleotide” are used interchangeably in thisapplication.

The term “solid support”, “support”, and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. See U.S. Pat. No.5,744,305 for exemplary substrates.

Multiplex Target Amplification Methods

In a first aspect, the methods disclosed herein provide a simple,multiplex targeted amplification method that does not require PCR. Foreach target to be amplified a circularization probe is synthesizedcontaining sequences that are complementary to sequences that flank thetarget to be amplified. In particular, the circularization probeincludes a first region that is complementary to a first sequence at the5′ end of the target and a second region that is complementary to asecond sequence at the 3′ end of the target, both regions of the probebeing complementary to the same strand of the target. Thecircularization probe is designed so that when the target strand ishybridized to the circularization probe the 5′ and 3′ flanking sequencesare brought together to facilitate joining of the ends of the strand toform a circularized target strand. The circularized target strandincludes the 5′ and 3′ flanking sequences and the target.

In some embodiments the circularization probe hybridizes to the targetso that flaps of non-target sequence are generated. The flaps can be 5′or 3′ overhangs. The flaps can be removed by enzymatic activity togenerate target ends for ligation to form circular target strands.

After circularization of the targets to be amplified, non circularizednucleic acids in the mixture can be digested using exonucleaseactivities and the circles can be amplified using rolling circleamplification (RCA). Primers for the RCA may be, for example, randomprimers, target specific primers, the circularization probes orfragments of the circularization probes. For a description of rollingcircle amplification see, for example, U.S. Pat. Nos. 6,183,960 and6,210,884 to Lizardi and U.S. Pat. No. 6,593,086 to Zhang. See also,Fire and Xu, “Rolling replication of short DNA circles”, Proc. Natl.Acad Sci. USA 92:4641-4645 (1995), Zhang, D Y, et al. Amplification oftarget-specific ligation-dependent circular probe. Gene, 211(2):277-285, 1998 and Lizardi, P M, et al., Mutation Detection andSingle-Molecule Counting Using Isothermal Rolling-Circle Amplification,Nature Genetics, 19: 225-232, 1998. See also Kumar U.S. Pat. No.5,912,124.

The presently disclosed methods are similar to those disclosed in Dahlet al. Nucleic Acids Res 33:e71 (2005) in that two sequence specificregions from the same strand of targeted DNA sequence separated by somedistance are used to facilitate formation of a single stranded circlefrom the target. However, in the method taught by Dahl et al., themethod incorporates universal priming sites into the circularized targetand the subsequent amplification is by PCR using primers to theuniversal priming sites. The method of Dahl et al. therefore requiresthe use of two oligonucleotides for each target, referred to as the“vector oligonucleotide” and the “selector probe”. The vectoroligonucleotide is about 34 bases in length and contains the universalpriming sites while the selector probe is longer (˜70 bases) andcontains the complement of the vector oligonucleotide flanked by targetspecific ends. The presently disclosed methods require a singleoligonucleotide similar to the selector probe but lacking the regionthat is the complement of the vector oligonucleotide. The presentlydisclosed methods therefore require one oligonucleotide per targetinstead of the two oligonucleotides required by the previous methods andthe target specific oligonucleotide is shorter (˜40 instead of ˜70bases) because no universal priming sites are required. Porreca et al.Nat Methods 4(11):931-6 (2007) also describes a method for multiplexamplification of a selected set of target sequences based on the padlockprobe methodology described in, for example, U.S. Pat. No. 5,871,921(Landegren et al.), Hardenbol et al. Nat. Biotechnol. 21, 673-678 (2003)and Hardenbol et al. Genome Res. 15, 269-275 (2005).

In the presently disclosed methods a single oligonucleotide of about 40bases may be used for each target to be amplified. In some embodimentsthe oligonucleotide may be shorter or longer, for example, 12 to 100bases. Because the target regions of the circularization probe arejuxtaposed and not separated by universal priming sites the sequencescan be selected to facilitate the use of a 5′ flap endonuclease,allowing the use of randomly sheared or fragmented genomic DNA. The endsof the fragments of the input nucleic acid need not be known.

In one embodiment the 5′ and 3′ target flanking regions are selected sothat they include common sequences of about 2 to 8 bases at the 5′ endof the 5′ target flanking region and at the 3′ end of the 3′ targetflanking region. After ligation, the targeted DNA is circularized, andthe uncircularized DNA, probes, and other nucleic acid with free endsmay be removed by treatment with specific exonucleases, such asexonuclease I, exonuclease VII, exonuclease III and T7 exonuclease. Thecircled DNAs are then amplified equally with random primers using DNApolymerase of high strand displacement such as Phi29. Commercialavailable kits such as TempliPhi from GE Healthcare can be used.Alternatively, the same probe pool can be used with Phi29 to produce theproducts. These multiplex amplified products can be used directly inmany applications, especially resequencing by tiling array orgenotyping.

A method of simple multiplex targeted amplification of genomic DNA, cDNAor RNA is described herein. In general there are four differentapproaches contemplated: (1) using DNA targets that have perfect ends bycleaving the DNA with one or more restriction enzymes and designing theprobes accordingly; (2) using targets that have a 5′ flap and a singlebase 3′ flap generated by cutting the DNA with one or more restrictionenzymes and designing the probes accordingly; (3) targeting the probe tohybridize so that the 5′ end is perfectly complementary to the probe andthe 3′ end hybridizes to generate a 3′ flap using DNA cut with one ormore restriction enzymes; and (4) targeting the probe to hybridize totwo regions flanking a region of interest and resulting in double flapsusing random sheared DNA (though restriction enzyme cut DNA can be usedalso).

A splint oligonucleotide or circularization probe is synthesizedcontaining two regions (T1′ and T2′ in FIG. 1) with length rangingpreferably from 6 to 60 bases and complementary to regions T1 and T2,respectively, of the genomic DNA or cDNA of the target species ofinterest. T1 and T2 are separated by a region that will be amplified.This region may be, for example, 50 to 500 bases or 500 to 5,000 basesor more than 5,000 bases). In preferred aspects the circularizationprobe length is about 40 bases (including ˜20 bases for each of T1 andT2). This length of oligo can be synthesized easily with high qualityeven without purification. A pool of these circularization probestargeting different regions of the DNA can be used to hybridize to thedesired targeted DNA, allowing the formation of circularized DNA afterligation (FIG. 1).

In FIG. 1 each of four different approaches are shown (A.-D.) In each,the DNA input is [101] and the circularization probe is [103]. In theembodiment shown in FIG. 1A the probe 103 hybridizes to the DNA 101 sothat the ends of the DNA are separated by at least a nick. The probe hasa 5′ region T1′ that is complementary to the 5′ end of the target 101and a 3′ region T2′ that is complementary to the 3′ end of the target.The ends of the target are known so the circularization probe isdesigned accordingly. T1′ and T2′ hybridize to the target so that theends of the target are juxtaposed. The nick can be closed by a ligationstep 105 to generate a closed circular DNA target 107. The closedcircular DNA target can then be amplified by RCA. In some aspects theremay be a gap between T1 and T2 upon hybridization of the circularizationprobe and the gap may be filed by a DNA polymerase.

In FIG. 1B the DNA 101 hybridizes to the probe 103 so that there is a 5′flap of two or more bases 109 and a 3′ flap of a single base 111. Thestructure is recognized by 5′ flap nucleases which catalyze removal ofthe 5′ flap and ligation of the end of the 3′ flap to the new 5′ end ofthe target strand generated by cleavage by the 5′ flap nuclease. Theresulting nick can be closed by ligation and the resulting circlesamplified.

In FIG. 1C the DNA 101 hybridizes to the probe 103 to generate a 3′ flapof two or more bases. The 5′ end of the target is of known sequence andthe 5′ end of the circularization probe is designed to be complementaryto the known 5′ end of the target. The 3′ end of the circularizationprobe is complementary to a region that is within the target. It is notnecessary to know the location of the 3′ end of the fragment. A 3′ flapnuclease is used to remove the 3′ flap. A DNA polymerase may be used toextend the 3′ end generated by the 3′ flap nuclease to create a gap tobe closed by ligase. The resulting circles can be amplified.

In FIG. 1D the DNA 101 hybridizes to the probe 103 to generate a 5′ flapof two or more bases 109 and a 3′ flap of two or more bases 119. The 5′flap nuclease removes the 5′ flap and a 3′ to 5′ exonuclease activity isused to remove the 3′ flap. A ligase is used to join the juxtaposed endsof the target that result. The circularized targets can be amplified.

For the embodiments shown in FIG. 1A both ends of the target are definedby restriction sites and the probe is designed to hybridize to becomplementary to regions flanking selected restriction sites. Becausethere is an optimal size range for the circle to be efficientlyamplified the choice of restriction enzymes may limit the targets thatare available for efficient amplification in a single reaction. To beefficiently amplified, target regions are selected to be flanked byselected restriction enzyme sites separated by a distance that is withinthe length that can be efficiently amplified. Preferably the targetfragments are a defined distance from the restriction sites bothupstream and downstream. For the embodiments shown in FIGS. 1B and Conly one end of the probe needs to be defined by a restriction site andthe second end can be selected for optimal circle size.

In a first embodiment, shown in FIG. 1A, the circularization probes maybe designed based on the restriction digestion so that once hybridized,a perfect nick will be formed (the 5′ and 3′ ends of the target will bejuxtaposed) and the nick can be sealed (in step 113) by formation of aphosphodiester bond (105) by DNA ligase, for example, T4 DNA ligase orTaq DNA ligase. After exonuclease treatment, the circularized DNAs 107can be amplified by rolling cycle amplification (RCA). In one aspect,the probe 103 may be used as a primer for RCA. Alternatively, randomprimers, a portion of the circularization probe or a target specificsequence outside of the region complementary to the circularizationprobe may be used as primers.

In another embodiment, shown in FIG. 1B, the circularization probes aredesigned so that when hybridized to the target a structure is generatedthat has a nick, a 5′ flap 109 and a 3′ flap of a single base 111. Astructure-specific flap nuclease may be used to remove the 5′ flap, forexample, the 5′ to 3′ exonuclease/endonuclease domain of the E coli DNApolymerase, Taq DNA polymerase or other eubacterial DNA polymerase, orthe Archeal or eukaryotic flap endonuclease 1 (FEN1), including humanFEN1 and Pfu FEN1. In a preferred embodiment, the circularization probemay be designed so that the 3′ end base of the 5′ flap is the same baseas the base of the 3′ flap. In step 115, a 5′ flap nuclease is used toremove the 5′ flap (109) and a ligase is added to ligate the ends of thetarget to form a circular target. The structure shown in the left handside of FIG. 1B may be referred to as a 5′ flap with 1 base 3′ flap.

In another embodiment, the probe hybridizes to the target so that theresulting structure has a 3′ flap 119 and no flap at the 5′ end, asshown in FIG. 1C, a 3′ flap nuclease may be used, such as the humanXeroderma pigmentosa complementation group F (XPF) (Sijbers A M et al.,Cell 86: 811-822, 1996), Archeal P. furiosus helicase-associatedendonuclease (Hef) (Komori et al., Genes Genet. Syst. 77: 227-241,2002), Archeal S. solfataricus XPF (Roberts J A et al., Mol. Microbiol.48: 361-371, 2003) and Nar71 (Guy C P et al., Nucleic Acids Research 32:6176-6186, 2004). See also, Matsui et al., J Biol Chem 274:18297-18309(1999) for a description of FEN1 in Pyrococcus horikoshii. The 3′ flapnucleases generally cuts a few sites near the flap junction (generatinga gap) and DNA polymerase may be used in step 121 to fill in the gap forligation of the ends. In a preferred aspect, less than all four dNTPsmay be included in the reaction, for example, only dATP and dGTP may beincluded and the probes are designed with a gap of only dA or dG after3′ flap nuclease treatment.

In the most flexible embodiment (shown in FIG. 1D), random sheared DNA,fragmented, for example, mechanically, chemically or enzymatically usingan enzyme such as DNase or Apyrase, may be hybridized to the probes togenerate primarily double flap structures as shown in FIG. 1D and alsoin FIG. 2. The random fragmentation will also generate some nick orgapped structures. The T1 and T2 sequences can be selected to flank thesequence of interest and preferably to have a common sequence at the 3′end of T2 and at the 5′ end of T1. It has been observed that cleavage byyeast FEN1 was more efficient at cleavage when the upstream primercontained a 1-nucleotide 3′-tail than when the upstream primer was fullyannealed (both contained a 5′ tail). See, Kao H-I et al., J. Biol. Chem.277: 14379-14389, 2002. Since 3′ flap nucleases can generate a gap andcan also cut nicking products, the preferred enzyme for step 123 is a 5′flap nuclease. A 3′ to 5′ exonuclease is also added to cleave the 3′flap and a ligase is used to close the nick. To allow the cleavage of 5′flap nuclease of the double flap structure the circularization probe isdesigned so that the two specific target regions (T1 and T2) areselected so the 3′-end sequence of one target region will overlap withthe 5′-end sequence of the other target region to create 2 to 8 bases ofidentical sequence. This shared sequence is present only once in theprobe but is present in each of the target sequences.

In the double flap example shown in FIG. 2 the overlapping region is the“TTGC” present in both SEQ ID NO. 2 and 3 and underlined. The probe (SEQID NO. 1) has the AACG complement, but only once. Second, singlestranded DNA-specific exonucleases such as exonuclease I, exonuclease Tor exonuclease VII may be added together with 5′ flap nucleases (e.g.,human FEN1). The 3′ to 5′ ssDNA exonucleases will degrade the ssDNA 3′flap, but most likely with some blunts, some 3′ extension of a few basesor even a few bases into the dsDNA. Therefore, with the few overlappingbases, the creation of 5′ flap with 1 base of 3′ flap is significantlyincreased in the presence of 3′ to 5′ exonucleases, allowing removal of5′ flap to create a perfect nick with high specificity and efficiency(FIG. 2). The nick may be ligated in the presence of DNA ligase. In apreferred embodiment, a computer program may be used to select the T1and T2 sequences so that they have overlapping end sequences.

As shown in FIG. 2, the probe, 5′-AGAGTCCTAT GGCTGGTTCA TGCAAGCTGCCGGAGTGAAC GCTGAGTGAG-3′ (SEQ ID NO. 1), has a 5′ targeting region 203and a 3′ targeting region 201 and a shared region 205. The 5′ targetingregion 203 is complementary to a first region 207 in the target and whenhybridized to the target 5′ overhang 209 is results. The 3′ targetingregion 201 is complementary to a second region 211 in the target andwhen hybridized to the target 3′ overhang 213 results.

In step 221 a 3′ to 5′ exonuclease is added and overhang 213 is digestedso that only a short flap remains (TTG is shown). The flap iscomplementary to the shared region 205 of the probe.

The complementarity of the 3′ end of SEQ ID No. 4 to the shared regionof the probe allows reversible branch migration in step 225 so that onlya single base at the 3′ end of SEQ ID No. 4 is unpaired (shown as a Gbase). In step 231, the flap nuclease cleaves between the G and C atpositions 16 and 17 of SEQ ID No. 3, cleaving SEQ ID No. 3 into twofragments 5′-GTGTTTGTGC GTTTTG-3′ (SEQ ID No. 5) and 5′-CATGAACCAGCCATAGGACT CT-3′ (SEQ ID No. 6), and leaving a nick (between the G atthe 3′ end of SEQ ID No. 4 and the C at the newly generated 5′ end ofSEQ ID No. 5. The nick can be closed by ligase. The resulting product233 is a close circular target 233 containing SEQ ID NO. 7.

The sequences shown in FIG. 2 are the exemplary products expected. SEQID No. 1 (5′-AGAGTCCTAT GGCTGGTTCA TGCAAGCTGC CGGAGTGAAC GCTGAGTGAG-3′)is the circularization probe for this target. SEQ ID No. 2(5′-CTCACTCAGC GTTCACTCCG GCAGCTTGCC TGTACTGTT-3′) is the sequence atthe 5′ end of the target with the 5′ portion being complementary to the3′ portion of SEQ ID No. 1 and the 3′ portion forming a 3′ flap. SEQ IDNO. 3 (5′-GTGTTTGTGC GTTTTGCATG AACCAGCCAT AGGACTCT-3′) is the sequenceat the 3′ end of the target with the 5′ portion forming a 5′ flap andthe 3′ portion being complementary to the 5′ portion of SEQ ID No. 1.SEQ ID NO. 4 (5′-CTCACTCAGC GTTCACTCCG GCAGCTTG-3′) is generated fromSEQ ID No. 2 after 3′ to 5′ exonuclease treatment. SEQ ID No. 5(5′-GTGTTTGTGC GTTTTG-3′) is the 5′ portion of SEQ ID No. 3 aftercleavage with flap nuclease and SEQ ID No. 6 (5′-CATGAACCAG CCATAGGACTCT-3′) is the 3′ portion of SEQ ID No. 3 after cleavage with flapnuclease. SEQ ID No. 7 (5′-CTCACTCAGC GTTCACTCCG CAGCTTGCAT GAACCAGCCATAGGACTCT-3′) is the 3′ and 5′ target flanking regions after ligationand includes the 5′ portion of SEQ ID No. 2 and the 3′ portion of SEQ IDNo. 3. The underlined section originated is from SEQ ID No. 2 and thebold section is from SEQ ID No. 3.

In on embodiment where a 5′ flap endonuclease is used a 5′ to 3′ ssDNAexonuclease, such as RecJ or Exo VII, may be used to shorten the lengthof 5′ flap. By doing this, the efficiency of the removal of long 5′flaps, for example, greater than 50 bases, may be increased. Removalefficiency has been shown to decrease with increased flap length,although very good cleavage can be obtained up to 500 bases in mostcases. The lengths of the flaps may be, for example, 1 to 500 bases or 1to 1,000 bases. The length of the targets to be amplified may be about100 to 5,000 bases, or about 100 to 10,000 bases. In some aspects eachtarget may be greater than 10,000 bases. Because RCA is being used foramplification and not PCR the length of the amplicons may be longer. Insome aspects that circularization probe may be selected to include arecognition site for a restriction endonuclease. After RCA theendonuclease may be used to cleave the RCA product into segments.

In another aspect Dna2 may be used to shorten the 5′ and 3′ flaps. SeeKim et al., Nucleic Acids Res. 34:1854-1864 (2006) and Stewart et al.JBC 281:38565-38572 (2006).

Once the targeted DNA is circularized by ligation, the DNAs other thanthe circled single stranded DNA may be removed by treatment withexonucleases, for example, exonuclease I, exonuclease VII, exonucleaseIII and T7 exonuclease.

In preferred aspects, the circularized product is amplified with minimalbias using Phi29 and random primers. Alternatively, the starting probepool can be used with Phi29 to produce the products. These multiplexamplified products can be used directly in many applications, especiallyresequencing by tiling array or genotyping.

1. In one aspect, the targets may be used for resequencing using theAffymetrix resequencing arrays, for example, the CUSTOMSEQ arrayproduct. Resequencing arrays allow variation detection in a sequence ofinterest by tiling probes for all possible single nucleotide variationswithin the sequence. They have been used in a number of studies. See,for example, Cutler et al., Genome Res. 11:1913-25 (2001), Lipkin etal., Nature Genet. 36:694-699 (2004), Zwick et al., Genome Biol 6:R10,(2005) and Warrington et al., Hum Mutat 19:402-9 (2002). Resequencingarrays are currently commercially available for analysis of up to 300 kbof double stranded DNA (600,000 bases total). Arrays can be purchasedfor 50 kb or 100 kb as well. Larger sequences can also be analyzed. Theamplification methods disclosed herein can be used in place of the longrange PCR amplification methods currently used for resequencing,eliminating the need to normalize and pool amplicons prior tohybridization. In some aspects kits for amplification to prepare targetsfor resequencing are disclosed. Preferred kits may have one or more ofthe following: at least 100 different dU probes, including one for eachtarget to be amplified, a ligase, UDG and a flap endonuclease, that maybe selected from a 5′ flap endonuclease and a 3′ flap endonuclease. Theamplification products may be fragmented and labeled by standard methodsprior to hybridization to arrays. Fragmentation may be, for example, byDNase treatment with end labeling using terminal transferase.

The non-PCR targeted multiplex amplification methods disclosed hereinare simple and highly specific. The embodiments shown in FIGS. 1A and Bare preferably used with targets that have known and defined ends, forexample, restriction fragments. As a result this method may be limitedsomewhat in the choice of the targets that can be amplified because theyrequire at least one defined end (defined 5′ and 3′ ends in 1A anddefined 3′ ends in 1B). Defined ends may be achieved, for example, bycleavage with one or more restriction enzymes. Although the requirementfor defined ends may make it difficult to amplify some targets usingthis approach, resulting in less than 100% coverage of a genome, choiceof enzymes may facilitate amplification of a majority of the targets ofinterest. Use of multiple enzymes may be used to further increase thecoverage of these embodiments.

The embodiment shown in FIG. 1C uses a 3′ flap nuclease to remove the 3′flap. The circularization probes are designed to be complementary to aregion at the 5′ end of the target and to include the 5′ end generatedby the fragmentation method, for example, if fragmentation is byrestriction digestion then the T1′ portion of the circularization probeis complementary to the region at the end of the restriction fragmentand generated by cleavage. The T2′ portion is not restricted to thenatural fragment end and can be selected to generate a desired circlelength. Preferably the 3′ flap is less than about 500 bases and morepreferably less than about 50 bases.

The double flap strategy shown in FIG. 1D is the most flexibleembodiment because it does not require specific ends to be generated inthe target by the initial cleavage so there are fewer limitations on thecleavage methods and the coverage can be 100%, similar to individualPCR. Optimization of the choice of flap nucleases and exonucleases andreaction conditions may be used to overcome the reduced efficiency thatis expected when removal of flaps at both ends is required.

In many aspects of the presently described methods a flap endonucleaseis used to remove overhanging ends prior to ligation of the ends of thetarget DNA. Flap endonucleoases (FEN-1) have been described in a numberof organisms including mouse, human, yeast and a number of thermophilicorganisms. They are structure-specific endonucleases that cleave 5′ flapstructures endonucleotyltically and have a double-strand-specific 5′-3′exonuclease activity. The exonuclease activity utilizes double-strandedDNA with a nick or gap, and the endonuclease activity requires a flapstructure. In prokaryotes the FEN 1 activity is the 5′ nuclease domainof DNA polymerase I. There is a separate polypeptide in eukaryotes,archaea and some bacteriophage. For additional information aboutdifferent enzymes and substrate specificities, see, Xu et al., J. Biol.Chem 276:30167-30177 (2001) and Kaiser et al. J Biol Chem274:21387-21394 (1999). FENs catalyze hydrolytic cleavage of thephosphodiester bond at the junction of single and double stranded DNA(see, Harrington and Lieber, EMBO 13:1235-46 (1994); Harrington andLieber, J Biol Chem 270:4503-8 (1995)). In cells, FEN-1 is one of theenzymes required for lagging strand DNA replication and in particular,the maturation of Okazaki fragments by generation of ligatable nicks.Flap endonuclease activities are used, for example, in the TAQMAN assayand in the INVADER assay. Taq DNA polymerase I endonucleolyticallycleaves DNA substrates, releasing unpaired 5′ arms of bifurcatedduplexes. See Lyamichev et al., PNAS 96:9143 (1999) and Lyamichev etal., Science 260: 778-783 (1993).

Targets amplified using the methods disclosed herein may be used for avariety of studies. In one aspect the targets contain known polymorphicregions and the amplified targets are analyzed to determine the genotypeof the sample at the polymorphic regions. In another aspect the methodsare combined with methods for analysis of methylation, for example, thetargets may be treated with bisulfite prior to amplification so thatmethylation dependent modifications are made to the sequence and thosechanges are maintained in the amplification product and can be detectedas changes in the sequence. Methylation detection using bisulfitemodification and target specific PCR have been disclosed, for example,in U.S. Pat. Nos. 5,786,146, 6,200,756, 6,143,504, 6,265,171, 6,251,594,6,331,393, and 6,596,493.

Prior to circularization, the nucleic acid sample containing the targetsmay be treated with bisulfite. During bisulfite treatment, unmethylatedcytosine is converted to uracil and methylated cytosine remainscytosine. See Clark et al., Nucleic Acids Res., 22(15):2990-7 (1994).When the modified strand is copied a G will be incorporated in theinterrogation position (opposite the C being interrogated) if the C wasmethylated and an A will be incorporated in the interrogation positionif the C was unmethylated. This results in a detectable sequencedifference between methylated and unmethylated positions.

Kits for DNA bisulfite modification are commercially available from, forexample, Human Genetic Signatures' Methyleasy and Chemicon's CpGenomeModification Kit. See also, WO04096825A1, which describes bisulfitemodification methods and Olek et al. Nuc. Acids Res. 24:5064-6 (1994),which discloses methods of performing bisulfite treatment and subsequentamplification on material embedded in agarose beads. In one aspect acatalyst such as diethylenetriamine may be used in conjunction withbisulfite treatment, see Komiyama and Oshima, Tetrahedron Letters35:8185-8188 (1994). See also, Hayatsu et al, Proc. Jpn. Acad. Ser. B80:189-194 (2004) for alternative procedures.

Bisulfite treatment allows the methylation status of cytosines to bedetected by a variety of methods. For example, any method that may beused to detect a SNP may be used, for examples, see Syvanen, Nature Rev.Gen. 2:930-942 (2001). Methods such as single base extension (SBE) maybe used or hybridization of sequence specific probes similar to allelespecific hybridization methods. In another aspect the MolecularInversion Probe (MIP) assay may be used. The MIP assay is described inHardenbol et al., Genome Res. 15:269-275 (2005) and in U.S. Pat. No.6,858,412.

The methods may also be used for analysis of copy number. See, forexample Wang et al., Nucleic Acids Res. 33:e183 (2005) and Ji et al.,Cancer Res. 66:7910-9 (2006). In other aspects the methods may be usedto analyze targets from compromised samples, for example, formaldehydefixed and paraffin embedded (FFPE) samples or degraded samples. In manyembodiments the amplification products may be analyzed by hybridizationto an array of probes. Preferred arrays include those commerciallyavailable from Affymetrix, Inc. and include tiling arrays, geneexpression arrays, mapping arrays (10K, 100K, 500K, and SNP 5.0),promoter arrays and tag arrays. The amplification products may also beanalyzed by hybridization to arrays of oligonucleotides attached tobeads or other solid supports.

In another aspect methods for multiplex target amplification using flapnucleases to generate specific ends for ligation of common primingsequences are disclosed. These methods are illustrated in FIG. 3 and arerelated to methods for probe-based multiplex PCR with common oligoaddition that have been previously described in U.S. Pat. No. 7,208,295and Faham M et al., PNAS 102: 14717-14722, (2005), each of which isincorporated herein by reference in its entirety for all purposes. Inthese methods pools of probes are used to capture and normalize thetargeted DNAs followed by the ligation of common oligos to the targets.The common oligos flank the targets and allow multiplex amplification ofthe targets using common primers to the common oligo sequences. Inpreferred aspects, the probes contain dU replacing at least some of thedT, allowing removal of the probes by treatment with UDG to generateabasic sites and cleavage at the abasic sites before amplification ofthe targets. The dU-containing probes can be synthesized chemically, butin preferred embodiments they are obtained by PCR amplification usingspecific primers and replacing dTTP with dUTP

The use of dU probes in 2-step multiplex PCR has been previouslydescribed in Faham M et al., PNAS 102: 14717-14722, (2005), but briefly,the method is as follows. In the first stage, multiple pairs of oligos(from 50-200 pairs) are added in the same tube and PCR is performed.Then different pools of 50 to 200-plex first stage products are pooledand used for capture or normalization with common oligos and ligase,followed by a second PCR with common primers. The second stage can be ina plex of about 1000 but can be over 10,000 (“plex” herein refers to thenumber of targets amplified in a single reaction, so 100 plex means thatabout 100 different targets are amplified in the reaction). The firststage PCR is with specific target primers for each target so the numberof amplifications that can be done in a single reaction (the plex level)is currently most efficient at lower levels, for example, less than 200,preferably less than 100 or less than 50.

In many embodiments, the dU probe includes a target specific regionflanked by common regions at both ends. The dU probes may be obtained,for example, by PCR with the inclusion of dUTP. The dUTP may replace thedTTP in the reaction or the reaction may include varying amounts of bothdUTP and dTTP. Individual dU probes are obtained and pooled together,preferentially in equal or approximately equal molar concentration. Thispool is then used in a capture reaction with genomic DNA or cDNA,together with two common oligonucleotides (C1 and C2) that arecomplementary to the common sequences in one strand of the dU probe.After ligation the strand that is formed has the structure C1-target-C2and can be amplified with primers designed to target the C1 and C2regions.

In some aspects, a unique tag sequence can be also included in a dUprobe. When a tag is included an oligonucleotide complementary to thetag may also be included. The probe may be designed so that the tagcomplement is included in the ligated product along with the target andflanked by the common oligonucleotides. See U.S. Pat. No. 7,208,295 fora discussion of how tag sequences may be included in dU probes and howthey may be used in subsequent detection methods. When the C1-tagcomplement-target-C2 is amplified the tag complement is amplified aswell.

The dU method does not require 2 stages of PCR and can be performedusing targeted genomic DNA or cDNA. It has been demonstrated to have asensitivity equivalent to or lower than 60 ng human genomic DNA (30zmol). In some aspects nucleases, such as single strand specificnucleases or mung bean nuclease, may be used to cleave flaps createdduring the hybridization of targeted DNA to the dU probes. The examplesincluded herein demonstrate methods for successful multiplex PCRamplification using genomic DNA directly.

In FIG. 3 each of four different four embodiments is shown (A-D). Ineach, the DNA input 301 contains the target 302 that contains a regionthat is complementary to the dU probe 303 for that target. Each has anenzymatic step (307, 317, 319 and 321) that includes ligation. Theproduct generated (shown to the right of horizontal arrows) is doublestranded and includes a top strand (306) that includes flankingoligonucleotides ligated to a target (junctions where ligation hasclosed a nick are shown by an X) and the input probe (303). The probecan then be separated from 306 and 306 can be amplified by PCR usingprimers to the common flanking sequences. The probe 303 may, forexample, contain deoxyuracil and can be fragmented by using UDG or UNGin combination with heat treatment or enzymatic cleavage with anendonuclease that recognizes abasic sites generated by UDG.

In the first embodiment, shown in FIG. 3A, the ends of the DNA are“perfect” used herein to indicate that the ends of the target 302 areknown and defined, for example, by cleavage with one or more restrictionenzymes or by amplification with target specific primers. The dU probe303 is designed so that the target 302 hybridizes to the probe so thatthere are no unpaired bases in the target. The oligonucleotides 304 and105 that are complementary to the common sequences at the ends of the dUprobe hybridize to the dU probe flanking the target so that the ends areadjacent, leaving a “nick” that can be closed by ligase in step 307.This embodiment may be referred to as the “nick” embodiment. Ligationbetween the 5′ end of 302 and the 3′ end of 304 and between the 3′ endof 302 and the 5′ end of 305 results in the single strand 306 thatincludes the target flanked by the common sequences 304 and 305. The dUprobe 303 may then be digested using UDG cleavage methods and 306 can beamplified by PCR using common primers.

In another embodiment, shown in FIG. 3B (“5′ flap with 1 base 3′ flap”)the dU probes may be designed to hybridize to targets so that one end ofthe target 302 forms a single stranded 5′ flap when the target ishybridized to the dU probe. Similarly, in the embodiment shown in FIG.3C the target hybridizes to the probe so that a 3′ flap is formed. Forthe embodiments shown in FIGS. 3B and C the genomic DNA may be cut withone or more restriction enzymes so the ends are known but bothembodiments allow greater flexibility in target selection than theembodiment shown in FIG. 3A because targets can be amplified from largerfragments that may not have been efficiently amplified without removalof a flap region.

In the embodiment shown in FIG. 3D the DNA can have both a 5′ flap and a3′ flap. This allows for use of DNA that has ends that may have beengenerated by random shearing although restriction enzyme cut DNA mayalso be used.

In the first embodiment (shown in FIG. 3A) the ends of the fragments areknown, and the probes are designed based on the known ends. In apreferred embodiment the ends are defined by restriction digestion. Oncehybridized, perfect nicks will be formed and can be sealed by DNA ligase(including but not limited to T4 DNA ligase and Taq DNA ligase). Thisembodiment is the simplest functionally (see FIG. 4 and FIG. 5 forresults of amplifications with perfect nicks), but is least flexible forselection of the targets. However, multiple cleavage enzymes can beused, either in separate cleavage reactions that are combined prior tohybridization of probes or in double digests, to increase the coverageof targets.

In the embodiment shown in FIG. 3B, the probes 303 may be designed sothat the 5′ end of target 302 forms a 5′ flap structure when hybridizedto 303. In a preferred embodiment the oligo 309 is designed so that itforms a single base 3′ flap when hybridized to 103. See, Kao H-I et al.,J. Biol. Chem. 277: 14379-14389, 2002. (FIG. 1B). A structure-specificflap nuclease may then be used to cleave the target at the positionshown by the arrow. In preferred embodiments the flap nuclease may be,for example, the 5′ to 3′ exonuclease/endonuclease domain of aeubacterial DNA polymerases such as E. coli DNA polymerase and Taq DNApolymerase (Lyamichev V. et al., Science 260: 778-783; Kaiser et al., J.Biol. Chem. 274: 21387-21394, 1999), or the archeal or eukaryotic flapendonuclease 1 (FEN1), including murine FEN1 (Harrington J J and LieberM R, EMBO J. 13: 1235-1246, 1994), yeast FEN1 (Harrington J J and LieberM R, Genes Dev. 8: 1344-1355, 1994), human FEN1 (Hiraoka L R et al.,Genomics 25: 220-225, 1995), P. horikoshii Pho FEN1 (Matsui E. et al.,J. Biol. Chem. 274: 18297-18309, 1999), or Pfu FEN1 (Kaiser et al., J.Biol. Chem. 274: 21387-21394, 1999). These enzymes typically require afree 5′ end and therefore, do not cleave at a mismatch or loop createdduring hybridization of dU probes and targeted sequences.

For maximal specificity and activity, the probe and the oligo to beligated to the 3′ end of the target after removal of the flap aredesigned so that the 3′-end base of the 5′ flap is the same base as thebase at the 3′ end of the oligo. This embodiment may be referred to as“double flap with 1 base 3′ flap”. As shown in FIG. 4 and example 1below, both the 5′ to 3′ exo/endo-nuclease domain of the Taq DNApolymerase and human FEN1 can efficiently remove the 5′ flap to createligatable nicks. As described below in the Examples, A 355-plexamplification using DdeI restriction enzyme-digested human genomic DNAwas performed and 100% of the targets were amplified successfully wherean amplification of a specific target was successful if the ratio ofmultiplex PCR product to dU probes for that target was greater than 0.1)(FIGS. 6-8).

Although any single restriction enzyme may allow less than 100% coverageof a genome (coverage of ˜85% of the human genome is obtained using DdeIrestriction enzyme if 70-300 bp PCR amplicons are targeted with 5′ flaplengths of up to 500 bases), use of two or three restriction enzymeseparately may be used to increase the coverage to greater than about85%, about 85 to 95% or about 95% to ˜99%.

In another embodiment the target hybridized to the probe so thestructure has a 3′ flap, as shown in (FIG. 3C) (“nick 3′ flap”). A 3′flap nuclease (also structure-specific) may be used, such as the humanxeroderma pigmentosa complementation group F (XPF), (Sijbers A M et al.,Cell 86: 811-822, 1996), Archeal P. furiosus helicase-associatedendonuclease (Hef, Komori et al., Genes Genet. Syst. 77: 227-241, 2002),Archeal IXPF (Roberts J A et al., Mol. Microbiol. 48: 361-371, 2003) andNar71 (Guy C P et al., Nucleic Acids Research 32: 6176-6186, 2004). Seealso, Matsui et al., J Biol Chem 274:18297-18309 (1999) for adescription of FEN1 in Pyrococcus horikoshii.

The 3′ flap nucleases may remove a few bases near the flap junction(generating a gap), and DNA polymerase and dNTPs may be used in step 319to fill in the gap. In preferred aspects, less than all four differenttypes of dNTPs may be included in the reaction (for example, only dATPand dCTP included) and the probes may be designed so that a gap of onlyone or two types of dNTP (for example, only dA or dC) is formed after 3′flap nuclease treatment.

In the most flexible embodiment (FIG. 3D), randomly fragmented DNA (forexample, mechanically sheared, or chemically or enzymatically fragmentedby an enzyme such as DNase or Apyrase) may be hybridized to the probesto generate flap structures at both ends (though some nick or gappedstructures may also be present in the randomly sheared DNA). For the 5′flap, the same 1 base 3′ flap in the oligo 309 is preferably included sothe 5′ flap nuclease such as human FEN1 can cut it efficiently andprecisely to create a nick for ligation in step 321. For the 3′ flap, a3′ flap nuclease such as XPF can be used simultaneously or sequentially.

In another embodiment double flap structures (as shown in FIG. 3D) areresolved without the use of a 3′ flap nuclease. Many 3′ flap nucleasescan generate a gap and can also cut nick products efficiently, requiringthat gaps be filled using a polymerase. This can be avoided by using a3′ to 5′ ssDNA exonuclease in combination with a 5′ flap nuclease,(instead of a 3′ flap endonuclease) to remove a 3′ flap in a double flaptarget.

In this embodiment, the probe is designed so the 3′-flap sequence nearthe junction will overlap with the 5′-end sequence of the nearby commonoligo to create 2 to 8 bases of sequence that is identical in the 3′flap and the common oligo (GG to GGCGCGCC in the common oligo, forexample). The repeated sequence is present at the junction betweentarget and common sequence only once in the dU probe. The identicalsequence does not have to be in the 5′-end of common oligo, but can bein the bases near the nick so a double flap structure will be created—a5′ flap from the end of the common oligo and the 3′ flap from thetarget. The 3′ to 5′ ssDNA exonucleases such as exonuclease I,exonuclease T or exonuclease VII will degrade the ssDNA 3′ flap, butwill generally leave some blunt ends but mostly 3′ extensions of a fewbases. Because there area few overlapping bases at the ends and the endswill “breathe” a structure that has a 5′ flap with 1 base of 3′ flapwill be generated at least some of the time, allowing removal of the 5′flap to create a perfect nick with high specificity and efficiency. Thenick may be ligated by DNA ligase. Without the 3′ to 5′ ssDNAexonucleases, 5′ flap nucleases such as FEN1, are unable to cut the 5′flap with a 3′ flap of more than 1 base (Kao H-I et al., J. Biol. Chem.277: 14379-14389, 2002).

In some embodiments, because genomic DNA or cDNA is used directly, theresulting amplification products will be proportionate to the startingcopy amount of individual targets, allowing the quantitation of copynumber of genomic DNA or expression level of cDNA. Also contemplated arecomputer implemented methods for selecting target sequences withoverlapping sequence regions with the common oligo.

The use of 5′ flap nucleases and 3′ to 5′ ssDNA exonucleases to createnick products from double flap structures may be combined with otherstrategies to provide a powerful strategy for multiplex targetedamplification. In one aspect the methods are applied to theselector-guided multiplex amplification of Dahl et al., Nucleic AcidsRes. 33: e71, 2005). In this method, a synthetic oligo with two specificsequence (from the same strand of DNA separated by ˜70-1000 bases, forexample) separated by a common sequence in the middle of the oligo isused. Random sheared genomic DNA may be hybridized to this oligo,creating a circle of the target after removal of the 5′ flaps and 3′flaps.

In another embodiment, where a 5′ flap nuclease is used, a 5′ to 3′ssDNA exonuclease, such as RecJ or Exo VII (which contains both 5′ to 3′and 3′ to 5′ exonuclease activities), may be used to shorten the lengthof the 5′ flap. By doing this, the efficiency of the removal of long 5′flaps (for example, greater than 50 bases) may be increased, since theremoval efficiency is dependent on flap length, although very goodcleavage can be obtained up to at least 500 bases in most cases. (SeeFIG. 7 and FIG. 8). In another aspect Dna2 may be used to shorten the 5′and 3′ flaps. See Kim et al., Nucleic Acids Res. 34:1854-1864 (2006) andStewart et al. JBC 281:38565-38572 (2006). The lengths of the flaps maybe, for example, 1 to 500 bases or 1 to 1,000 bases. The length of thetargets to be amplified may be about 100 to 1,000 or 2,000 bases, butthe targets may be longer, for example, 2,000 to 10,000 baspairs.

Once the targeted DNAs are captured and ligated to common oligos, the dUprobes may be degraded with uracil-DNA glycosylase (UDG or UNG) plusheat treatment or treatment with an AP endonuclease, and the targetedDNAs may be amplified with common PCR primers.

Several aspects of the multiplex PCR process described below could alsobe used to increase the sensitivity and efficiency and make the assayeasier and more robust. For example, since many other proteins interactwith 5′ or 3′ flap nucleases, the efficiency of flap removal or activityof the flap nucleases can be increased by addition of one or moreadjunct proteins. One such proteins is proliferating cell nuclearantigen (PCNA), which has been demonstrated to increase the activity ofhuman FEN1 by up to 50 fold. Furthermore, Sso PCNA is required for the3′ flap removal activity of Sso XPF. FIG. 5 shows that using Sso XPF inthe presence of PCNA functions to remove a 3′ flap of 40 or 500 bases.

In another embodiment a fused oligo, (C1C2) consisting of both commonsequences C1 and C2 as a single contiguous oligo, may be used. Afterhybridization, enzymatic treatment and ligation, the specific targetedsequences will be circularized with the fused C1C2. This approach allowsthe removal of the DNA that hasn't been circularized (for example, theoriginal targeted DNA and the probes) by treatment with DNA exonuclease.Exonucleases that may be used include, for example, exonuclease I,exonuclease VII, exonuclease III and T7 exonuclease. The circularsequence can be opened with UDG if one or more dU is included in thecommon C1C2 oligo while the dU probes are also degraded, and amplifiedwith common PCR primers. Alternatively, circular DNAs can be amplifiedwith minimal bias using Phi29 and random primers or a primer to C1C2.Rolling circle amplification (RCA) may be used. For a description ofrolling circle amplification see, for example, U.S. Pat. Nos. 6,183,960and 6,210,884 to Lizardi and U.S. Pat. No. 6,593,086 to Zhang. See also,Fire and Xu, Proc. Natl. Acad Sci. USA 92:4641-4645 (1995), Zhang, D Y,et al., Gene, 211(2): 277-285, 1998 and Lizardi, P M, et al., NatureGenetics, 19: 225-232, 1998. See also Kumar U.S. Pat. No. 5,912,124.

Commercial available kits containing phi 29 enzymes and random primerpools can be used. Alternatively, the same probe pool can be used withPhi29 to produce the products. These multiplex amplified products can beused directly in many applications, especially resequencing by tilingarray or genotyping.

Circular double-stranded DNA may not form efficiently for smaller sizes,although as small as 70 bp “mini plasmids” have been reported. Thecircularization method may also be of reduced efficiency due to theformation of linear structures instead of circularized structures.Efficiency may be improved by denaturing the double stranded linear dUprobes and making circular ssDNA dU probes using CIRCLIGASE™ ssDNAligase from EpiCentre Biotechnologies (Madison, Wis.). The circularssDNA dU probes may be used to capture targeted DNA.

In one embodiment solid-phase dU capture is used instead ofsolution-based capture. For example, one of the dU probe strands can belinked to biotin and can be immobilized to a tube coated withstreptavidin or to magnetic beads coupled with streptavidin. Onelimitation is the space restriction created which could lead to lowerefficiency. The solid-phase capture allows the removal and exchange ofthe buffer/reagents conveniently without desalting or purificationsteps.

To minimize the unwanted cleavage of nick structures by 5′ flapnucleases and 3′ flap nucleases before ligation, the 5′-end or 3′-end ofthe common oligos used in capture may include one or morephosphorothioate bonds. The phosphorothioate bond is resistant to mostexonucleases (Putney S D et al., Proc. Natl. Acad. Sci. USA 78:7350-7354, 1981; Skerra A, Nucleic Acids Res. 20: 3551-3554, 1992) andpotentially the structure-specific flap nucleases also.

The key advantages of the disclosed methods of multiplex PCR are thatthey have high sensitivity and specificity and can be scaled up toamplify larger number of targets, for example, more than 10,000-plex,50,000-plex or 100,000-plex. It has been demonstrated that 3 ng humangenomic DNA (˜1000 copies) can be used to amplify the amplicons with100% success rate (ratio of >0.1, FIG. 7). Similar amounts of genomicDNA, based on TAQMAN™ real time PCR quantitation (Applied Biosystems),from formalin fixed paraffin embedded tissue (FFPE) was also amplifiedequally well (FIG. 8), opening up an opportunity to selectively amplifythese and other degraded DNA efficiently and in highly multiplexedreactions (100 to 100,000-plex). These multiplex amplified products canbe used directly in many applications, especially for downstreammismatch repair detection (MRD) or resequencing by tiling array orgenotyping. MRD is disclosed in U.S. Pat. Nos. 7,153,652, 6,709,827 and6,406,847, which are incorporated herein by reference for all purposes.In one embodiment the methods may be used for the preparation of targetsused in resequencing analysis using GENECHIP CUSTOMSEQ® arrays.

In some embodiments kits for multiplex amplification are disclosed. Kitsmay include, for example, reagents and arrays for resequencing orgenotyping applications. The kits may include, for example, dU probes, aflap endonuclease, a DNA polymerase, common oligonucleotides that mayinclude nuclease resistant linkages, primers for PCR, buffers, andcontrol DNA. The kits may also include arrays and instructions forperforming one or more of the methods disclosed herein. For each targetto be amplified a different dU probe may be included. The kit mayinclude, for example, 100, 500, 1,000, 2,000 or 10,000 to 500, 1,000,2,000, 10,000, 20,000, 50,000 or 100,000 different dU probes.

In the examples below the multiplex strategy with 5′ flap nuclease hasbeen shown to work very well with 355-plex and should be scalable tomuch higher levels of multiplexing (1,000 to 100,000 or higher).Employing a strategy that uses a 5′ flap nuclease only has thelimitation that less than 100% of an entire genome can be targetedbecause of the requirement for use of restriction enzymes. Embodimentsthat use random sheared genomic DNA with both 5′ and 3′ flaps may beused to obtain 100% coverage and high flexibility in designing theprobes. The methods do require generation of dU probes corresponding tothe targets, but once the dU probe is made, it can be re-amplified togenerate large amounts of stable probe that can be used for manymultiplex PCR. In general, the dU probe need be synthesized andamplified only once.

In one aspect target sequences from a nucleic acid sample are amplifiedby fragmenting the nucleic acid sample to obtain fragments; adding tothe fragments a plurality of dU probes to the complex mixture, whereinthere is a dU probe for each target sequence and wherein each dU probecomprises: (i) a central target region that is complementary to a targetsequence; (ii) a 5′ first common sequence; (iii) a 3′ second commonsequence. Adding to the mixture a first oligonucleotide that iscomplementary to the first common sequence and a second oligonucleotidethat is complementary to the second common sequence. Adding a 5′ flapnuclease, at least one species of dNTP, a 3′ flap nuclease, a DNA ligaseand a DNA polymerase to the mixture of (b) to form targets ligated tothe first and second common oligonucleotides. Adding a uracil DNAglycosylase to the mixture above and cleaving the dU probes. Thenamplifying the targets ligated to the first and second commonoligonucleotides using primers to the first and second common sequences.

In one aspect, a plurality of nucleic acid targets of distinct sequenceare amplified. In a first step a first oligonucleotide and a secondoligonucleotide are appended to each of the nucleic acid targets byannealing each target and a respective first oligonucleotide to arespective probe from a plurality of probes in a reaction mixture,wherein each probe comprises a first region of complementarity to arespective one of said targets, a first oligo positioning regiondirectly adjacent thereto, the nucleotide of the templatecomplementarity region and the nucleotide of the first oligo positioningregion that are directly adjacent within said probe being firstjunctional nucleotides that define a first probe junction, and a secondoligo positioning region directly adjacent to the first templatecomplementarity region, the nucleotide of the template complementarityregion and the nucleotide of the second oligo positioning region thatare directly adjacent within said probe being second junctionalnucleotides that define a second probe junction, and wherein each saidfirst oligonucleotides includes a first common priming sequence and aregion that is complementary to the first oligonucleotide positioningregion of the probe. Then creating a first ligatable free end at thenucleotide of each template that is annealed to the junctionalnucleotide of its respective probe's first target complementarityregion, wherein each of said ligatable free ends is created by removingtarget regions that are noncomplementary to said probe firstcomplementarity region by a flap endonuclease. The next step is ligatingeach first oligonucleotide to its respective target first free end toappend the first oligonucleotide to its respective target within theplurality of nucleic acid targets and appending a second oligonucleotideto each of the plurality of targets of distinct sequence by: annealing arespective second oligonucleotide to each probe concurrently withannealing of the target to the probe, wherein the second oligonucleotideincludes a terminal region that is complementary to the secondoligonucleotide positioning region of its respective probe and a secondcommon sequence, the terminal nucleotide of said terminaloligonucleotide region being annealed to the junctional nucleotide ofthe probe's second oligonucleotide positioning region. The next step iscreating a second ligatable free end at the target nucleotide that isannealed to the second junctional nucleotide of the probe's first targetcomplementarity region; and then ligating the second oligonucleotide tothe target second free end. The next step is separating the targets,after ligation, from the probes and the oligonucleotides and thenamplifying a region of each target by PCR using primers to the commonsequence.

In one aspect, the amplified targets may be used for resequencing usingthe Affymetrix resequencing arrays, for example, the CUSTOMSEQ™resequencing array. Resequencing arrays allow variation detection in asequence of interest by tiling probes for all possible single nucleotidevariations within the sequence. They have been used in a number ofstudies. See, for example, Cutler et al., Genome Res. 11:1913-25 (2001),Lipkin et al., Nature Genet. 36:694-699 (2004), Zwick et al., GenomeBiol 6:R10, (2005) and Warrington et al., Hum Mutat 19:402-9 (2002).Resequencing arrays are currently commercially available for analysis ofup to 300 kb of double stranded DNA (600,000 bases total). Arrays can bepurchased for 50 kb or 100 kb as well. Larger sequences can also beanalyzed. The amplification methods disclosed herein can be used inplace of the long range PCR amplification methods currently used forresequencing, eliminating the need to normalize and pool amplicons priorto hybridization. In some aspects kits for amplification to preparetargets for resequencing are disclosed. The amplification products maybe fragmented and labeled by standard methods prior to hybridization toarrays.

In many aspects of the presently described methods a flap endonucleaseis used to remove overhanging ends prior to ligation of the ends of thetarget DNA. Flap endonucleoases (FEN-1) have been described in a numberof organisms including mouse, human, yeast and a number of thermophilicorganisms. They are structure-specific endonucleases that cleave 5′ flapstructures endonucleotyltically and have a double-strand-specific 5′-3′exonuclease activity. The exonuclease activity utilizes double-strandedDNA with a nick or gap, and the endonuclease activity requires a flapstructure. In prokaryotes the FEN 1 activity is the 5′ nuclease domainof DNA polymerase I. There is a separate polypeptide in eukaryotes,archaea and some bacteriophage. For additional information aboutdifferent enzymes and substrate specificities, see, Xu et al., J. Biol.Chem 276:30167-30177 (2001) and Kaiser et al. J Biol Chem274:21387-21394 (1999). FENs catalyze hydrolytic cleavage of thephosphodiester bond at the junction of single and double stranded DNA(see, Harrington and Lieber, EMBO 13:1235-46 (1994); Harrington andLieber, J Biol Chem 270:4503-8 (1995)). In cells, FEN-1 is one of theenzymes required for lagging strand DNA replication and in particular,the maturation of Okazaki fragments by generation of ligatable nicks.Flap endonuclease activities are used, for example, in the TAQMAN assayand in the INVADER assay. Taq DNA polymerase I endonucleolyticallycleaves DNA substrates, releasing unpaired 5′ arms of bifurcatedduplexes. See Lyamichev et al., PNAS 96:9143 (1999) and Lyamichev etal., Science 260: 778-783 (1993).

The targets may be selected for analysis of a variety of genetic andepigenetic features. The target regions may contain known polymorphicregions and the amplified targets are analyzed to determine the genotypeof the sample at the polymorphic regions. In another aspect the methodsare combined with methods for analysis of methylation, for example, thetargets may be treated with bisulfite prior to amplification so thatmethylation dependent modifications are made to the sequence and thosechanges are maintained in the amplification product and can be detectedas changes in the sequence. Methylation detection using bisulfitemodification and target specific PCR have been disclosed, for example,in U.S. Pat. Nos. 5,786,146, 6,200,756, 6,143,504, 6,265,171, 6,251,594,6,331,393, and 6,596,493.

Prior to hybridization to the dU probes, the nucleic acid samplecontaining the targets may be treated with bisulfite. During bisulfitetreatment, unmethylated cytosine is converted to uracil and methylatedcytosine remains cytosine. See Clark et al., Nucleic Acids Res.,22(15):2990-7 (1994). When the modified strand is copied a G will beincorporated in the interrogation position (opposite the C beinginterrogated) if the C was methylated and an A will be incorporated inthe interrogation position if the C was unmethylated. This results in adetectable sequence difference between methylated and unmethylatedpositions. The methods disclosed herein may be combined with the methodsdisclosed in U.S. application Ser. No. 11/923,649 filed Oct. 24, 2007.

Kits for DNA bisulfite modification are commercially available, forexample, Human Genetic Signatures' Methyleasy and Chemicon's CpGenomeModification Kit. See also, WO04096825A1, which describes bisulfitemodification methods and Olek et al. Nuc. Acids Res. 24:5064-6 (1994),which discloses methods of performing bisulfite treatment and subsequentamplification on material embedded in agarose beads. In one aspect acatalyst such as diethylenetriamine may be used in conjunction withbisulfite treatment, see Komiyama and Oshima, Tetrahedron Letters35:8185-8188 (1994). See also, Hayatsu et al, Proc. Jpn. Acad. Ser. B80:189-194 (2004) for alternative procedures.

Bisulfite treatment allows the methylation status of cytosines to bedetected by a variety of methods. For example, any method that may beused to detect a SNP may be used, for examples, see Syvanen, Nature Rev.Gen. 2:930-942 (2001). Methods such as single base extension (SBE) maybe used or hybridization of sequence specific probes similar to allelespecific hybridization methods. In another aspect the MolecularInversion Probe (MIP) assay may be used. The MIP assay is described inHardenbol et al., Genome Res. 15:269-275 (2005) and in U.S. Pat. No.6,858,412.

The methods may also be used for analysis of copy number. See, forexample Wang et al., Nucleic Acids Res. 33:e183 (2005) and Ji et al.,Cancer Res. 66:7910-9 (2006). In other aspects the methods may be usedto analyze targets from compromised samples, for example, formaldehydefixed and paraffin embedded (FFPE) samples or degraded samples. In manyembodiments the amplification products may be analyzed by hybridizationto an array of probes. Preferred arrays include those commerciallyavailable from Affymetrix, Inc. and include tiling arrays, geneexpression arrays, mapping arrays (10K, 100K, 500K, SNP 5.0 and SNP6.0), promoter arrays and tag arrays. The amplification products mayalso be analyzed by hybridization to arrays of oligonucleotides attachedto beads or other solid supports.

In another aspect the amplification products may be analyzed bysequencing methods such as those marked by Roche/454, Helicose,Illumina/Solexa, and ABI (SOLID) and others.

EXAMPLES Example 1

Taq Polymerase and FEN1 specifically cleave the 5′ flap to create nicksfor ligation. A control dU probe and corresponding targets withdifferent flap length (0, 50 or 500 bases) were generated by PCR andpurified. 50 amol of each were hybridized for about 3 hours in 30 mMTris-HCl, pH 7.9, 15 mM MgCl₂, 0.1% BSA by step-wise annealing (70° C.40 min, 65° C. 40 min, 60° C. 40 min, 55° C. 20 min, 50° C. 20 min andthen 4° C. hold after denaturation at 94° C. for 5 min. The reaction wasdesalted, the 5′ flap was removed and the targets were ligated using 40U Taq ligase plus either 5 U Taq DNA polymerase, 0.25 μl hFEN1 or 0.25μl Pfu FEN1 or none. After ligation, the reactions in the upper panelwere purified by QIAQUICK™ spin columns (QIAGEN). All ligation productswere treated with UDG, then amplified with common primers. An aliquot ofeach was separated by gel electrophoresis as shown in FIG. 4.

The expected position of migration of the expected product is shown byan arrow in both the upper and lower panels. DNA ladders are shown inlanes 1, 18, and 19. Negative controls of herring sperm DNA are shown inlanes 2, 3, 10, 11, 20, 21, 28 and 29. Lanes 2 to 9 were treated withTaq polymerase and Taq ligase, lanes 10 to 17 were treated with Taqligase only, lanes 20 to 27 were treated with 0.25 μl hFEN1 and ligaseand lanes 28 to 35 were treated with 0.25 μl PfuFEN1 and ligase. Asexpected, a product of the expected length is observed in lanes 4-9, 12,13, 22-27 and 30-35. The probe generated structures in lanes 14-17 aredesigned to have 5′ overhangs of 50 (14 and 15) or 500 bases (16 and 17)and are not expected to result in amplifiable product in the absence ofa 5′ flap nuclease. As expected no amplified product is observed inthese lanes or in the negative control lanes with just herring spermDNA. Lanes 4, 5, 12, 13, 22, 23, 30 and 31 have probes, targets andoligos that hybridize to generate nicks that are ligatable withoutcleavage of 5′ flaps so product is observed in all of these lanes, evenin lanes 12 and 13 that do not have added 5′ flap nuclease.

Example 2

FIG. 5 shows that a 3′ flap structure as shown in FIG. 3C can be removedby Sso XPF/PCNA mix (kindly provided by Dr. Malcolm White, UK) so thatthe common oligos can be ligated to the ends of the target and thetarget amplified by PCR. The reaction conditions were those used inExample 1 except different 3′ flap lengths were tested with 0.25 μl SsoXPF/PCNA, 40 U Taq DNA ligase, 2.5 U Taq DNA polymerase, 300 μM each ofdATP and dCTP (the 5 bases next to the ligation site in the 3′ flapposition) for the upper panel. The reactions in the lower panel had 0.25μl Sso XPF/PCNA, 40 U Taq DNA ligase, 2.5 U Taq DNA polymerase, andvarying amounts of dATP and dCTP as indicated. The template was eitherherring sperm DNA (HS), a target without 5′ or 3′ flaps (perfect), atarget with a 3′ flap of 40 bases (3′+40) or a target with a 3′ flap of500 bases (3′+500). The amplification product is not observed in thelane where there is no added dATP/dCTP and the 3′+500 lane. This islikely because the 3′ flap endonuclease removes some of the bases nearthe nick generating a gap that can be filled in by DNA polymerase in thepresence of the appropriate dNTPs. The structure of the probe-targetcomplex is analogous to that shown in FIG. 3C.

Example 3

FIG. 6 Specific amplification of 355 targets simultaneously from normalcontrol genomic DNA (Promega) and an FFPE human genomic DNA. Genomic DNAwas cut with DdeI and 3 ng-100 ng was used in direct multiplex PCR witha dU probe pool (50 amol each) in 20 μl volume. To prepare the dU probepool individual PCR reactions, in the presence of dUTP, were performedfor each of 384 targets and successful products were obtained for 370 ofthe 384 (in subsequent studies PCR amplification conditions that allowsuccessful amplification of the remaining 14 have been identified). 369of the dU probes were used for capture (one was excluded because itcontained a highly repetitive Alu sequence. The dU probes and digestedDNA were mixed in a hybridization reaction with the common oligos firstto allow hybridization of the dU probes, the targets and the oligos andto allow flap formation. The FEN1 and ligase were added after thehybridization step. These steps may also be combined if a thermostableFEN1 is used and denaturation, annealing, flap removal and ligation canbe cycled. After the ligation and flap removal the dU probes aredegraded using uracil-DNA glycosylase followed by heating at 95° C. Thenthe targets are amplified by PCR using the common primers. The far leftlane and far right lane are 100 bp markers (NEB). The specific PCR sizesare from 145-375 bp. Notice that no background was seen in 100 ng yeastRNA which was used as a carrier except a nonspecific band below 100 bp.

FIG. 7 shows the results of array analysis of dU-based mPCR productsfrom FIG. 6. Either 0.5 μl (1.5 U) or 2 μl (6 U) of human FEN1(Trevigen) was used for 3 or 100 ng human genomic DNA (Promega) digestedwith DdeI. Panels A and C are 3 ng human genomic DNA, 0.5 μl FEN1, B andD are 100 ng DNA and 2 μl FEN1. Panels C and D show flap size on the Xaxis and ratio of the signal from the DdeI fragmented DNA with FEN to acontrol dU amplification on the y-axis (control is the starting dU probepool fragmented, labeled and hybridized to a copy of the same array).The ratio was greater than 0.1 for all targets in both C and Dindicating 100% success for the amplification. After amplification, thePCR products were purified, fragmented and end labeled. The labeledfragments were hybridized to a custom resequencing array(Kinaser520379_(—)7G) according to the Custom Resequencing protocol. ThedU probe pool was fragmented, labeled and hybridized to the array as acontrol.

FIG. 8 shows comparison of the targets amplified from FFPE DNA in FIG. 6to the amplification products from human genomic DNA (also from FIG. 6).The results show that efficient mPCR amplification can be obtained using0.5 μl human FEN1 and 3 ng DNA from an FFPE sample. Each target wasamplified from the FFPE samples with about the same signal as from thenormal DNA (panels A and B). All of the targets were amplified from boththe 3 ng and 10 ng FFPE samples with a ratio to dU1114 that was greaterthan 0.1 (see panels C and D). The samples are those shown in FIG. 6.

Additional genotyping methods are disclosed in Wang H-Y et al. (2005)Genome Res. 15: 276-283 and Faham M. et al. (2005) Proc. Natl. Acad.Sci. USA 102: 14712-14722. Multiplex amplification methods that can beused in combination with the disclosed methods are described in Dahl etal. (2005), Shapero et al., (2001) Genome Res. 11: 1926-1934, Shapero etal. (2004) Nucleic Acids Res. 32: e181 and Pemov et al. (2005) NucleicAcids Res. 33: e11.

Yeast flap endonuclease is described in Kao H I et al. (2002). J. Biol.Chem. 277: 14379-14389. A method for using the 5′ to 3′ exonucleaseactivity of Taq DNA polymerase is described in Holland P M et al. (1991)Proc. Natl. Acad. Sci. USA 88: 7276-7280.

Nucleases that may be used in combination with the methods disclosedherein are described in Guy et al (2004) Nucleic Acids Res. 32:6176-6186, Nishino et al. (2006) 16: 60-67, Matsui et al. (2004) J.Biol. Chem. 279: 16687-16696, Komori et al. (2002) Genes Genet. Syst.77: 227-241, Roberts et al., (2003) Mol. Microbiol. 48: 361-371, SijbersA M et al. (1996) Cell 86: 811-822, Hiraoka L R et al. (1995) Genomics25: 220-225, Harrington J J and Lieber M R (1995) J. Biol. Chem. 270:4503-4508, Harrington J J and Lieber M R (1994) EMBO J. 13: 1235-1246(endonuclease), Kaiser M W et al. (1999) Biol. Chem. 274: 21387-21394(exonuclease), Lyamichev et al. (1993) Science 260: 778-783(endonuclease), and Liu et al., (2004) Annu Rev. Biochem. 73: 589-615(Flap endo 1).

CONCLUSION

It is to be understood that the above description is intended to beillustrative and not restrictive. Many variations of the invention willbe apparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should be determined withreference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. All cited references,including patent and non-patent literature, are incorporated herewith byreference in their entireties for all purposes as if each had beenindividually and specifically incorporated by reference herein.

We claim:
 1. A method for amplifying a plurality of target sequencesfrom a complex mixture of nucleic acid comprising: (a) fragmenting thenucleic acid to obtain a fragmented nucleic acid sample; (b) adding aplurality of circularization probes to the fragmented nucleic acidsample to form a mixture, wherein there is a circularization probe foreach target sequence in the plurality and wherein each circularizationprobe comprises: (i) a 5′ target region that is perfectly complementaryto a first region in a complementary target sequence, (ii) a 3′ targetregion that is perfectly complementary to a second region in thecomplementary target sequence and (ii) a central region that isperfectly complementary to a third region in the complementary targetsequence and also perfectly complementary to a fourth region in thecomplementary target sequence wherein said third and fourth regions aredistinct regions but share a common sequence of at least 2 contiguousbases; (c) adding a 3′ to 5′ exonuclease to the mixture; (d) adding a 5′flap nuclease to the mixture; (e) adding a ligase to the mixture,following step (d), to generate circularized target sequences; and (f)amplifying the circularized target sequences by rolling circleamplification.
 2. The method of claim 1 wherein the common sequenceshared by the third region and the fourth region is between 2 and 8bases in length.
 3. The method of claim 1 wherein the rolling circleamplification is primed by the circularization probes.
 4. The method ofclaim 1 wherein the 3′ to 5′ exonuclease is selected from the groupconsisting of exonuclease I, exonuclease VII, exonuclease III and T7exonuclease.
 5. The method of claim 1 wherein the flap endonuclease isFEN1 nuclease.
 6. The method of claim 1 wherein the flap endonuclease isTaq DNA polymerase I.
 7. A method for amplifying a plurality of targetsequences from a nucleic acid sample comprising: (a) fragmenting thenucleic acid sample by a method that generates fragments that terminateat the 5′ end of one strand with a known sequence; (b) adding aplurality of circularization probes to the fragments from (a), whereineach circularization probe is specific for a target in the plurality oftarget sequences and wherein each circularization probe comprises (i) afirst 5′ region that is complementary to a first sequence in the targetwherein said first sequence comprises at least 15 bases of the knownsequence at the 5′ end of the target and includes the 5′ end of thetarget and (ii) a second 3′ region that is complementary to a secondsequence in the target that is 3′ of the first sequence, is at least 15bases and is separated from the first sequence by a third sequence,wherein said circularization probe hybridizes to said target to form astructure wherein the first sequence and the second sequence are broughtinto juxtaposition by hybridization of the circularization probe and a3′ single stranded flap sequence comprising the 3′ end of the targetstrand is generated; (c) adding a ligase, a 3′ flap nuclease, a DNApolymerase and at least one species of dNTP to form closed circles fromsaid targets; and (d) amplifying said closed circles.
 8. The method ofclaim 7 wherein said amplification is by rolling circle amplification,wherein a Phi29 DNA polymerase is used and the primer is selected fromrandom primers, target specific primers and primers that include thejunction created by circularization.
 9. The method of claim 8 whereinsaid plurality of target sequences comprises at least 1000 differentgenomic sequences.
 10. A method for amplifying a plurality of targetsequences from a nucleic acid sample comprising: (a) fragmenting thenucleic acid sample by a method that generates fragments of knownsequence at the 3′ end of the target strands to be amplified; (b) addinga plurality of circularization probes to the fragments, wherein theplurality comprises a different circularization probe for each targetsequence to be amplified and wherein each circularization probecomprises: (i) a first 5′ region that is complementary to a firstsequence in the target; (ii) a second 3′ region that is complementary toa second sequence in the target, wherein said second sequence is 3′ ofsaid first sequence in the target strand to be amplified and hybridizesto the known sequence at the 3′ end of the target strand to be amplifiedand either includes the base at the 3′ end of the target strand orhybridizes so that the base at the 3′ end of the target forms a flap ofa single base; wherein said circularization probe hybridized to thetarget strand to form a structure wherein the first sequence and thesecond sequence are brought into juxtaposition by hybridization of thecircularization probe and a 5′ single stranded flap sequence comprisingthe 5′ end of the target strand is generated; (c) adding a 5′ flapnuclease and a ligase to form close circles from said targets; and (d)amplifying said closed circles.
 11. The method of claim 10 wherein thestructure comprises a 3′ flap of a single base and a 5′ flap of two ormore bases.
 12. The method of claim 10 wherein the 5′ flap endonucleaseis selected from the group consisting of a FEN1 nuclease and a Taq DNAPolymerase.
 13. A method for amplifying a plurality of target sequencesfrom a complex mixture of nucleic acid comprising: (a) fragmenting thenucleic acid to obtain a fragmented nucleic acid sample; (b) mixing thefragmented nucleic acid sample with a plurality of dU probes to thefragmented nucleic acid sample, wherein there is a dU probe for eachtarget sequence in the plurality and wherein each dU probe comprises:(i) a central target region that is perfectly complementary to a targetregion in a target fragment in the nucleic acid sample, wherein thetarget fragment comprises a target sequence and a 5′ non-target sequenceand wherein the target fragment hybridizes to the dU probe so that thetarget sequence is hybridized to the dU probe and the 5′ non-targetsequences forms a single stranded 5′ flap structure; (ii) a 5′ firstcommon sequence; and (iii) a 3′ second common sequence; (c) mixing thefragmented nucleic acid sample with a first oligonucleotide that iscomplementary to said first common sequence and a second oligonucleotidethat is complementary to said second common sequence; (d) adding a 5′flap nuclease to the mixture from (c) to cleave the 5′ flap from thetarget fragment to generate a new 5′ end in the target; (e) adding aligase to the reaction to ligate the first oligonucleotide to the new 5′end in the target and the second oligonucleotide to the 3′ end of thetarget; (f) cleaving the dU probes; and (g) amplifying the targetsequences using primers for said first and second common sequences. 14.The method of claim 13 wherein the target sequence comprises a sharedsequence of 2 to 8 bases and wherein said second oligonucleotide alsocomprises said shared sequence.
 15. The method of claim 13 furthercomprising adding a 3′ to 5′ exonuclease to said mixture and whereinsaid exonuclease is selected from the group consisting of exonuclease I,exonuclease VII, exonuclease III and T7 exonuclease.
 16. The method ofclaim 13 further comprising adding a 3′ flap endonuclease.
 17. Themethod of claim 13 wherein the 5′ flap endonuclease is selected from thegroup consisting of FEN1 nuclease and Taq DNA polymerase I.
 18. A methodfor amplifying a plurality of at least 100 target sequences from anucleic acid sample comprising: (a) fragmenting the nucleic acid sampleby a method that generates fragments that terminate at the 5′ end of onestrand with a known sequence; (b) adding a plurality of dU probes to thefragments from (a), wherein each dU probe is specific for a target inthe plurality of target sequences and wherein each dU probe comprises(i) a first 5′ region that is complementary to a first sequence in thetarget wherein said first sequence comprises at least 15 bases of theknown sequence at the 5′ end of the target and includes the 5′ end ofthe target and (ii) a second 3′ region that is complementary to a secondsequence in the target that is 3′ of the first sequence, is at least 15bases and is separated from the first sequence by a third sequence,wherein said dU probe hybridizes to said target to form a structurewherein the first sequence and the second sequence are brought intojuxtaposition by hybridization of the dU probe and a 3′ single strandedflap sequence comprising the 3′ end of the target strand is generated;(c) adding a ligase, a 3′ flap nuclease, a DNA polymerase and at leastone species of dNTP to form targets ligated to the first and secondoligonucleotides; (d) digesting the dU probes; and (d) amplifying thetargets ligated to the first and second oligonucleotides.
 19. The methodof claim 18 wherein said amplification is by rolling circleamplification, wherein a Phi29 DNA polymerase is used and the primer isselected from random primers, and target specific primers.
 20. A methodfor amplifying a plurality of target sequences from a nucleic acidsample comprising: (a) fragmenting the nucleic acid sample by a methodthat generates fragments of known sequence at the 3′ end of the targetstrands to be amplified; (b) adding a plurality of dU probes to thefragments, wherein the plurality comprises a different dU probe for eachtarget sequence to be amplified and wherein each dU probe comprises: (i)a first 5′ region that is complementary to a first sequence in thetarget; (ii) a second 3′ region that is complementary to a secondsequence in the target, wherein said second sequence is 3′ of said firstsequence in the target strand to be amplified and hybridizes to theknown sequence at the 3′ end of the target strand to be amplified andeither includes the base at the 3′ end of the target strand orhybridizes so that the base at the 3′ end of the target forms a flap ofa single base; wherein said dU probe hybridized to the target strand toform a structure wherein the first sequence and the second sequence arebrought into juxtaposition by hybridization of the dU probe and a 5′single stranded flap sequence comprising the 5′ end of the target strandis generated; (c) adding a 5′ flap nuclease and a ligase to form targetsligated to the first and second oligonucleotides; (d) digesting the dUprobes; and (e) amplifying the targets ligated to the first and secondoligonucleotides.
 21. The method of claim 20 wherein the structurecomprises a 3′ flap of a single base and a 5′ flap of two or more basesand wherein the 5′ flap endonuclease is selected from the groupconsisting of FEN1 and Taq DNA Polymerase I.